Gene regulation IIIBiochemistry 302Types of cis-elements that control transcription initiation in eukaryotesBasic principles underlying transcriptional regulation (i.e. activation) in eukaryotesExample: Network of TF interactions governing SM ?-actin gene transcriptionParadigm of transcriptional activation: Recruitment of Pol II GTF machineryStructural features of eukaryotic transcriptional regulators (modularity)Activators do other important things besides recruit RNA Pol II complexHows and whens of activator-mediated chromatin remodeling and PIC assemblyThermodynamics and chemistry underlying protein-DNA interactionZinc finger motif: first described by A. Klug and coworkers in 1985An example of three C (Cys)2H (His)2 zinc finger protein…..Transcriptional regulators use common DNA-binding motifsCommonly occurring motifs involved in DNA recognitionConsequences of forming heterodimers (e.g. C4 zinc-finger, bZIP, HLH proteins)Structure and function of “activation” (transcriptional regulatory) domainsOther mechanisms of gene regulation (its not all about transcription initiation)Gene regulation IIIBiochemistry 302Bob Kelm March 3, 2004Types of cis-elements that control transcription initiation in eukaryotes• Core promoter elements (for positioning RNA Pol II)– TATA box (TATAa/tAa/t, – 25 to –35)– Inr, Pyr-rich Initiator element (PyPyANa/tPyPy, +1)– DPE, Downstream Promoter Element (+30, yeast)• Activator/Repressor elements (regulated transcription)– Promoter-proximal elements (e.g. CpG islands, –100 to –200)– Distal enhancer/repressor elements– Insulator/boundary elementsfrom Lodish et al., Molecular Cell Biology, 3rdeditionRNA splicing does occur in yeast too.Basic principles underlying transcriptional regulation (i.e. activation) in eukaryotes• Assembly of a preinitiationcomplex is the key control point but…..• DNA must be freed from the nucleosome by chromatin remodeling factors. • Trans-acting factors (TFs) bind to cis-acting regulatory sequences and recruit the RNA Pol II GTF machinery.• TF binding sites may be close to or far away from transcription start site.• Multiple TFs cooperateto control transcription from a single promoter.Carey, M. Cell 92:5-8, 1998Example: Network of TF interactions governing SM α-actin gene transcriptionKuman, M. S and Owens, G. K. Arterioscler Thromb Vasc Biol. 23:737-747, 2003Paradigm of transcriptional activation: Recruitment of Pol II GTF machinery• Activators function by recruiting components of the basal transcription machinery to a promoter.• Direct protein-protein interaction between TF activation domains and components of the GTF machinery are necessary.– Reported Kds range from 10−4to 10−7M.– Network of contactsamong multiple activators & subunits of the RNA PolII GTF machinery result in transcriptional synergy.Parabolic responseSigmoidal responseK = e∆G/−RTCarey, M. Cell 92:5-8, 1998Structural features of eukaryotic transcriptional regulators (modularity)• Ordered DNA-binding domain• Pseudo-ordered “activation” domain– Modulate protein-protein interaction– Some TFs possess multiple activation domains• Flexible intervening region– Separate function domains– Protease sensitive• Consequences of modularity (creation of novel proteins)– Swapping of DNA-binding and activation domains– Research and drug development toolor repressorLodish et al. Molecular Cell Biology 3rdeditionActivators do other important things besides recruit RNA Pol II complexOrphanides, G. et al. (1996) Genes & Development 10:2657-2683Hows and whens of activator-mediated chromatin remodeling and PIC assemblyBefore Yeast HO gene promoterHuman IFN-βgene promoterHuman α1-AT gene promoterDuringAfterelongationC. J. Fry and C. L. Peterson (2002) Science 295:1847-1848Thermodynamics and chemistry underlying protein-DNA interaction• General themes− Steric (shape) and electrostatic (charge) compatibility of protein and DNA− Entropy driven: binding displaces counter ions & water− High affinity (Kd~ 10−9Mor less)• Direct Interactions− Specific recognition of individual bps by H-bonding between amino acids and functional groups− van der Waals contacts to thymine methyl groups − Electrostatic contacts to phosphates• Indirect Interactions− Bridging through water molecules− Conformational changes in protein/DNA to promote complementarity (e.g. TBP)Zinc finger motif: first described by A. Klug and coworkers in 1985• Inferred by amino acid sequence analysis of Xenopus laevis TFIIIA• Structure of first “zinc finger” of Zif268 is shown on the right– Two antiparallel β strands followed by an α helix, folding stabilized by coordination of a zinc ion – Zinc ion is chelated by 2 histidines (from α helix) and 2 cysteines (one from β strand, one from loop)FromFromPavletichPavletich, N.P. and Sabo, C.O., N.P. and Sabo, C.O.Science, 252,Science, 252,809809--817, 1991817, 1991An example of three C (Cys)2H (His)2zinc finger protein…..……hugging the major grooveN4 of C N7 & O6 of GRefined structure from C. Pabo lab Elrod-Erickson, M et al. Structure4, 1171-1180, 1996Transcriptional regulators use common DNA-binding motifs• Helix-turn-helix– Prokaryotic repressors and activators– Eukaryotic homeodomain proteins• Zinc-finger proteins– C2H2class bind as monomers (TFIIIA, Zif268)– C4(steroid receptors) and C6(Gal4) classes bind as dimers • Winged-helix (forkhead)– Bind as monomers– Histone H5, HNF3, and TFs involved in early development• Leucine zipper/bZIP– Bind as dimers– GCN4, Fos, Jun, and other related members• Helix-loop-helix– Bind as dimers (basic N-term α helix, loop region, C-term amphipathic αhelix– c-myc, Max, Mad, and others• Emerging motifs– β-sheet motifs − NFκB and MetRCommonly occurring motifs involved in DNA recognitionZinc fingerH-T-HbZIPLeucine (GCN4)Fig. 28.23Homeodomain, EngrailedC2H2Zn finger, Zif268C4Zn finger, glucocorticoid receptor (monomer only)C6Zn finger, yeast Gal4C6Zn finger, yeast Gal4Winged helix or forkhead, HNF3Leucine bZip, yeast Gcn4Leucine bZip, yeast Gcn4H-L-H + Leu zipper, MAXLodish et al.Molecular Cell Biology 3rdedition with citations thereinCoiled coil motifConsequences of forming heterodimers (e.g. C4zinc-finger, bZIP, HLH proteins)• Expansion of the number of potential DNA targets that a class of factors can bind.• Different combinations of activation domains are brought together.•